Baculovirus are a diverse group of viruses having a host range which is restricted to insects. They are large viruses having a lipid envelope and containing a genome of double-stranded circular DNA. Recombinant baculovirus (rBV) vectors, primarily derived from the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), are commonly used in industry and academia for the high-level production of heterologous proteins in insect cells. This expression system involves the infection of cultured insect cells with rBV into which the gene encoding the protein to be expressed has been inserted. See O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual. (Freeman, New York) for review of the baculovirus expression system.
Another potential use of rBV has recently appeared. It is demonstrated that rBV can transfer genetic material into mammalian cells, with a preference for hepatocytes. As long as a gene of interest is preceded by a promoter that is active in mammalian cells, the gene will be efficiently expressed in the target mammalian cells. See Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P., and Strauss, M. (1995), "Efficient gene transfer into human hepatocytes by baculovirus vectors", Proc. Natl. Acad. Sci. USA 92, 10099-10103; Boyce, F. M. and Bucher, N. L. R. (1996) "Baculovirus-mediated gene transfer into mammalian cells", Proc. Natl. Acad. Sci. USA 93, 2348-2352, incorporated herein by reference. This previously unobserved characteristic makes rBV potentially useful in human gene therapy. Recombinant baculovirus has several potential advantages for gene therapy. These include:
1. a very large DNA insert capacity PA1 2. A fairly high viral titer PA1 3. absence of a pre-existing immune response in humans PA1 4. lack of replication in mammals PA1 5. lack of toxicity in mammals PA1 6. lack of expression of viral genes in mammalian cells due to the insect-specificity of the baculovirus transcriptional promoters, and, therefore, a potential absence of a cytotoxic T lymphocyte response directed against these viral proteins
Nevertheless, there exists a need for a method to concentrate rBV to a very high titer. This will be required for in vivo gene therapy applications of high doses of rBV. This virus must be at high titer and must be present in a physiological buffer. Also, a purification/concentration step is critical in large scale in vitro production of proteins in insect cells. In order to produce proteins at very high levels, the insect cells must be infected with rBV at a high multiplicity of infection (moi). If the rBV has not been previously concentrated, but is rather simply the conditioned medium from the insect cells producing the virus, the volume of the inoculum would be a significant proportion of the total bioreactor volume. This may not be desirable, nor in some cases even possible, in large-scale manufacturing.
Viruses are conventionally concentrated to high titer by a combination of pelleting the virus through use of an ultracentrifuge and by banding the virus in, for instance, a sucrose gradient. See, for example, Sandig, V., Hofmann, C., Steinert, S., Jennings, G., Schiag, P., and Strauss, M. (1996) "Gene transfer into hepatocytes and human liver tissue by baculovirus vectors", Human Gene Therapy 7: 1937-1945 and Bowles, N. E., Eisensmith, R. C., Mohuiddin, R., Pyron, M. And Woo, S. L. C. (1996) "A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo", Human Gene Therapy 7: 1735-1742. Unfortunately, rBV preparations made in this manner tend to be badly aggregated (See Example 1). Therefore, a concentration step is needed that avoids this problem, but such a step has not heretofore been available.